Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 72
Filtrar
1.
Blood ; 143(16): 1670-1675, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38427750

RESUMO

ABSTRACT: Activated protein C (APC) was shown to release extracellular vesicles (EVs). APC bound to the EVs was thought to be responsible for cytoprotection. Our study demonstrates that the cytoprotective effects of APC-released EVs are independent of APC. APC-released EVs carry anti-inflammatory microRNAs in their cargo.


Assuntos
Citoproteção , Vesículas Extracelulares , Proteína C , Comunicação Celular , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Proteína C/metabolismo , Humanos
2.
Blood ; 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38820589

RESUMO

Chronic kidney disease (CKD) is a major contributor to morbidity and mortality in sickle cell disease (SCD). Anemia, induced by chronic persistent hemolysis, is associated with progressive deterioration of renal health resulting in CKD. Moreover, patients with SCD experience acute kidney injury (AKI), a risk factor for CKD, often during vasoocclusive crisis associated with acute intravascular hemolysis. However, the mechanisms of the hemolysis-driven pathogenesis of the AKI-to-CKD transition in SCD remain elusive. Here, we investigated the role of increased renovascular rarefaction and the resulting substantial loss of vascular endothelial protein C receptor (EPCR) on the progressive deterioration of renal function in transgenic SCD mice. Multiple hemolytic events raised circulating levels of soluble EPCR (sEPCR) indicating loss of EPCR from the cell surface. Using bone marrow transplantation and super-resolution ultrasound imaging, we demonstrated that SCD mice overexpressing EPCR were protective against heme-induced CKD development. In a cohort of SCD patients, plasma sEPCR was significantly higher in individuals with CKD than in those without CKD. This study concludes that multiple hemolytic events may trigger CKD in SCD through the gradual loss of renovascular EPCR. Thus, restoration of EPCR may be a therapeutic target, and plasma sEPCR can be developed as a prognostic marker for sickle CKD.

3.
Blood ; 139(1): 118-133, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34469511

RESUMO

Coagulation protease, factor VIIa (FVIIa), binds to endothelial cell protein C receptor (EPCR) and induces anti-inflammatory and endothelial barrier protective responses via protease-activated receptor-1 (PAR1)-mediated, biased signaling. Our recent studies had shown that the FVIIa-EPCR-PAR1 axis induces the release of extracellular vesicles (EVs) from endothelial cells. In the present study, we investigated the mechanism of FVIIa release of endothelial EVs (EEVs) and the contribution of FVIIa-released EEVs to anti-inflammatory and vascular barrier protective effects, in both in vitro and in vivo models. Multiple signaling pathways regulated FVIIa release of EVs from endothelial cells, but the ROCK-dependent pathway appeared to be a major mechanism. FVIIa-released EEVs were enriched with anti-inflammatory microRNAs (miRs), mostly miR10a. FVIIa-released EEVs were taken up readily by monocytes/macrophages and endothelial cells. The uptake of FVIIa-released EEVs by monocytes conferred anti-inflammatory phenotype to monocytes, whereas EEV uptake by endothelial cells resulted in barrier protection. In additional experiments, EEV-mediated delivery of miR10a to monocytes downregulated the expression of TAK1 and activation of the NF-κB-mediated inflammatory pathway. In in vivo experiments, administration of FVIIa-released EEVs to wild-type mice attenuated LPS-induced increased inflammatory cytokines in plasma and vascular leakage into vital tissues. The incorporation of anti-miR10a into FVIIa-released EEVs diminished the ability of FVIIa-released EEVs to confer cytoprotective effects. Administration of the ROCK inhibitor Y27632, which significantly inhibits FVIIa release of EEVs into the circulation, to mice attenuated the cytoprotective effects of FVIIa. Overall, our study revealed novel insights into how FVIIa induces cytoprotective effects and communicates with various cell types.


Assuntos
Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Fator VIIa/metabolismo , Inflamação/metabolismo , MicroRNAs/metabolismo , Animais , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Células THP-1
4.
Blood ; 140(13): 1549-1564, 2022 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-35895897

RESUMO

Deep vein thrombosis (DVT) is the third most common cause of cardiovascular mortality. Several studies suggest that DVT occurs at the intersection of dysregulated inflammation and coagulation upon activation of inflammasome and secretion of interleukin 1ß (IL-1ß) in restricted venous flow conditions. Our recent studies showed a signaling adapter protein, Gab2 (Grb2-associated binder 2), plays a crucial role in propagating inflammatory signaling triggered by IL-1ß and other inflammatory mediators in endothelial cells. The present study shows that Gab2 facilitates the assembly of the CBM (CARMA3 [CARD recruited membrane-associated guanylate kinase protein 3]-BCL-10 [B-cell lymphoma 10]-MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) signalosome, which mediates the activation of Rho and NF-κB in endothelial cells. Gene silencing of Gab2 or MALT1, the effector signaling molecule in the CBM signalosome, or pharmacological inhibition of MALT1 with a specific inhibitor, mepazine, significantly reduced IL-1ß-induced Rho-dependent exocytosis of P-selectin and von Willebrand factor (VWF) and the subsequent adhesion of neutrophils to endothelial cells. MALT1 inhibition also reduced IL-1ß-induced NF-κB-dependent expression of tissue factor and vascular cell adhesion molecule 1. Consistent with the in vitro data, Gab2 deficiency or pharmacological inhibition of MALT1 suppressed the accumulation of monocytes and neutrophils at the injury site and attenuated venous thrombosis induced by the inferior vena cava ligation-induced stenosis or stasis in mice. Overall, our data reveal a previously unrecognized role of the Gab2-MALT1 axis in thromboinflammation. Targeting the Gab2-MALT1 axis with MALT1 inhibitors may become an effective strategy to treat DVT by suppressing thromboinflammation without inducing bleeding complications.


Assuntos
Trombose , Trombose Venosa , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Células Endoteliais/metabolismo , Guanilato Quinases/metabolismo , Inflamassomos/metabolismo , Inflamação , Mediadores da Inflamação , Interleucina-1beta/metabolismo , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , NF-kappa B/metabolismo , Selectina-P/metabolismo , Tromboinflamação , Tromboplastina/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Trombose Venosa/genética , Fator de von Willebrand/metabolismo
5.
Blood ; 139(18): 2830-2841, 2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35143636

RESUMO

Recurrent spontaneous or trauma-related bleeding into joints in hemophilia leads to hemophilic arthropathy (HA), a debilitating joint disease. Treatment of HA consists of preventing joint bleeding by clotting factor replacement, and in extreme cases, orthopedic surgery. We recently showed that administration of endothelial cell protein C receptor (EPCR) blocking monoclonal antibodies (mAb) markedly reduced the severity of HA in factor VIII (FVIII)-/- mice. EPCR blocking inhibits activated protein C (APC) generation and EPCR-dependent APC signaling. The present study was aimed to define the role of inhibition of APC anticoagulant activity, APC signaling, or both in suppressing HA. FVIII-/- mice were treated with a single dose of isotype control mAb, MPC1609 mAb, that inhibits anticoagulant, and signaling properties of APC, or MAPC1591 mAb that only blocks the anticoagulant activity of APC. Joint bleeding was induced by needle puncture injury. HA was evaluated by monitoring joint bleeding, change in joint diameter, and histopathological analysis of joint tissue sections for synovial hypertrophy, macrophage infiltration, neoangiogenesis, cartilage degeneration, and chondrocyte apoptosis. No significant differences were observed between MPC1609 and MAPC1591 in inhibiting APC anticoagulant activity in vitro and equally effective in correcting acute bleeding induced by the saphenous vein incision in FVIII-/- mice. Administration of MAPC1591, and not MPC1609, markedly reduced the severity of HA. MAPC1591 inhibited joint bleed-induced inflammatory cytokine interleukin-6 expression and vascular leakage in joints, whereas MPC1609 had no significant effect. Our data show that an mAb that selectively inhibits APC's anticoagulant activity without compromising its cytoprotective signaling offers a therapeutic potential alternative to treat HA.


Assuntos
Artrite , Hemofilia A , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticoagulantes/farmacologia , Anticoagulantes/uso terapêutico , Receptor de Proteína C Endotelial , Hemartrose/tratamento farmacológico , Hemartrose/patologia , Hemartrose/prevenção & controle , Hemofilia A/complicações , Hemofilia A/tratamento farmacológico , Hemorragia , Camundongos , Proteína C/metabolismo
6.
Arterioscler Thromb Vasc Biol ; 43(1): 64-78, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36412194

RESUMO

BACKGROUND: Our recent studies suggest that sphingomyelin levels in the plasma membrane influence TF (tissue factor) procoagulant activity. The current study was performed to investigate how alterations to sphingomyelin metabolic pathway would affect TF procoagulant activity and thereby affect hemostatic and thrombotic processes. METHODS: Macrophages and endothelial cells were transfected with specific siRNAs or infected with adenoviral vectors to alter sphingomyelin levels in the membrane. TF activity was measured in factor X activation assay. Saphenous vein incision-induced bleeding and the inferior vena cava ligation-induced flow restriction mouse models were used to evaluate hemostasis and thrombosis, respectively. RESULTS: Overexpression of SMS (sphingomyelin synthase) 1 or SMS2 in human monocyte-derived macrophages suppresses ATP-stimulated TF procoagulant activity, whereas silencing SMS1 or SMS2 increases the basal cell surface TF activity to the same level as of ATP-decrypted TF activity. Consistent with the concept that sphingomyelin metabolism influences TF procoagulant activity, silencing of acid sphingomyelinase or neutral sphingomyelinase 2 or 3 attenuates ATP-induced enhanced TF procoagulant activity in macrophages and endothelial cells. Niemann-Pick disease fibroblasts with a higher concentration of sphingomyelin exhibited lower TF activity compared with wild-type fibroblasts. In vivo studies revealed that LPS+ATP-induced TF activity and thrombin generation were attenuated in ASMase-/- mice, while their levels were increased in SMS2-/- mice. Further studies revealed that acid sphingomyelinase deficiency leads to impaired hemostasis, whereas SMS2 deficiency increases thrombotic risk. CONCLUSIONS: Overall, our data indicate that alterations in sphingomyelin metabolism would influence TF procoagulant activity and affect hemostatic and thrombotic processes.


Assuntos
Hemostáticos , Trombose , Camundongos , Humanos , Animais , Esfingomielinas , Esfingomielina Fosfodiesterase/genética , Células Endoteliais/metabolismo , Trombose/genética , Hemostasia , Trifosfato de Adenosina
7.
Blood ; 138(4): 344-349, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34075401

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with the hypercoagulable state. Tissue factor (TF) is the primary cellular initiator of coagulation. Most of the TF expressed on cell surfaces remains cryptic. Sphingomyelin (SM) is responsible for maintaining TF in the encrypted state, and hydrolysis of SM by acid sphingomyelinase (ASMase) increases TF activity. ASMase was shown to play a role in virus infection biology. In the present study, we investigated the role of ASMase in SARS-CoV-2 infection-induced TF procoagulant activity. Infection of human monocyte-derived macrophages (MDMs) with SARS-CoV-2 spike protein pseudovirus (SARS-CoV-2-SP-PV) markedly increased TF procoagulant activity at the cell surface and released TF+ extracellular vesicles. The pseudovirus infection did not increase either TF protein expression or phosphatidylserine externalization. SARS-CoV-2-SP-PV infection induced the translocation of ASMase to the outer leaflet of the plasma membrane, which led to the hydrolysis of SM in the membrane. Pharmacologic inhibitors or genetic silencing of ASMase attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Inhibition of the SARS-CoV-2 receptor, angiotensin-converting enzyme-2, attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Overall, our data suggest that SARS-CoV-2 infection activates the coagulation by decrypting TF through activation of ASMase. Our data suggest that the US Food and Drug Administration-approved functional inhibitors of ASMase may help treat hypercoagulability in patients with COVID-19.


Assuntos
COVID-19/sangue , Macrófagos/virologia , Proteínas de Membrana/fisiologia , SARS-CoV-2 , Esfingomielina Fosfodiesterase/fisiologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Trombofilia/etiologia , Tromboplastina/fisiologia , Enzima de Conversão de Angiotensina 2/fisiologia , COVID-19/complicações , Micropartículas Derivadas de Células , Ativação Enzimática , Humanos , Hidrólise , Macrófagos/enzimologia , Terapia de Alvo Molecular , Plasmídeos , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores Virais/fisiologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielinas/fisiologia , Trombofilia/sangue , Trombofilia/tratamento farmacológico , Trombofilia/enzimologia
8.
Blood ; 137(24): 3428-3442, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33534910

RESUMO

Recombinant factor FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type (WT), EPCR-overexpressing, and PAR1-R46Q-mutant mice, but not EPCR-deficient or PAR1-R41Q-mutant mice. In vivo studies revealed that administration of FVIIa to WT, EPCR-overexpressing, and PAR1-R46Q-mutant mice, but not EPCR-deficient or PAR1-R41Q-mutant mice, increased the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.


Assuntos
Endotélio Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Fator VIIa/farmacologia , Hemostasia/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Receptor PAR-1/metabolismo , Substituição de Aminoácidos , Animais , Vesículas Extracelulares/genética , Hemostasia/genética , Humanos , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Receptor PAR-1/genética , Proteínas Recombinantes/farmacologia
9.
Blood ; 135(25): 2211-2223, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32294155

RESUMO

We recently showed that clotting factor VIIa (FVIIa) binding to endothelial cell protein C receptor (EPCR) induces anti-inflammatory signaling and protects vascular barrier integrity. Inflammation and vascular permeability are thought to be major contributors to the development of hemophilic arthropathy following hemarthrosis. The present study was designed to investigate the potential influence of FVIIa interaction with EPCR in the pathogenesis of hemophilic arthropathy and its treatment with recombinant FVIIa (rFVIIa). For this, we first generated hemophilia A (FVIII-/-) mice lacking EPCR (EPCR-/-FVIII-/-) or overexpressing EPCR (EPCR++ FVIII-/-). Joint bleeding was induced in FVIII-/-, EPCR-/-FVIII-/-, and EPCR++FVIII-/- mice by needle puncture injury. Hemophilic synovitis was evaluated by monitoring joint bleeding, change in joint diameter, and histopathological analysis of joint tissue sections. EPCR deficiency in FVIII-/- mice significantly reduced the severity of hemophilic synovitis. EPCR deficiency attenuated the elaboration of interleukin-6, infiltration of macrophages, and neoangiogenesis in the synovium following hemarthrosis. A single dose of rFVIIa was sufficient to fully prevent the development of milder hemophilic synovitis in EPCR-/-FVIII-/- mice. The development of hemophilic arthropathy in EPCR-overexpressing FVIII-/- mice did not significantly differ from that of FVIII-/- mice, and 3 doses of rFVIIa partly protected against hemophilic synovitis in these mice. Consistent with the data that EPCR deficiency protects against developing hemophilic arthropathy, administration of a single dose of EPCR-blocking monoclonal antibodies markedly reduced hemophilic synovitis in FVIII-/- mice subjected to joint bleeding. The present data indicate that EPCR could be an attractive new target to prevent joint damage in hemophilia patients.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Receptor de Proteína C Endotelial/deficiência , Hemartrose/prevenção & controle , Hemofilia A/complicações , Animais , Anticorpos Monoclonais/farmacologia , Citocinas/fisiologia , Receptor de Proteína C Endotelial/antagonistas & inibidores , Receptor de Proteína C Endotelial/imunologia , Receptor de Proteína C Endotelial/fisiologia , Fator VIIa/uso terapêutico , Hemartrose/tratamento farmacológico , Hemartrose/etiologia , Hemartrose/fisiopatologia , Hemofilia A/tratamento farmacológico , Hemofilia A/genética , Camundongos , Camundongos Knockout , Punções/efeitos adversos , Ratos , Proteínas Recombinantes/uso terapêutico , Sinovite/etiologia , Sinovite/prevenção & controle
11.
Arterioscler Thromb Vasc Biol ; 41(1): 250-265, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33028097

RESUMO

OBJECTIVE: TF (Tissue factor) plays a key role in hemostasis, but an aberrant expression of TF leads to thrombosis. The objective of the present study is to investigate the effect of 4-hydroxy-2-nonenal (HNE), the most stable and major oxidant produced in various disease conditions, on the release of TF+ microvesicles into the circulation, identify the source of TF+ microvesicles origin, and assess their effect on intravascular coagulation and inflammation. Approach and Results: C57BL/6J mice were administered with HNE intraperitoneally, and the release of TF+ microvesicles into circulation was evaluated using coagulation assays and nanoparticle tracking analysis. Various cell-specific markers were used to identify the cellular source of TF+ microvesicles. Vascular permeability was analyzed by the extravasation of Evans blue dye or fluorescein dextran. HNE administration to mice markedly increased the levels of TF+ microvesicles and thrombin generation in the circulation. HNE administration also increased the number of neutrophils in the lungs and elevated the levels of inflammatory cytokines in plasma. Administration of an anti-TF antibody blocked not only HNE-induced thrombin generation but also HNE-induced inflammation. Confocal microscopy and immunoblotting studies showed that HNE does not induce TF expression either in vascular endothelium or circulating monocytes. Microvesicles harvested from HNE-administered mice stained positively with CD248 and α-smooth muscle actin, the markers that are specific to perivascular cells. HNE was found to destabilize endothelial cell barrier integrity. CONCLUSIONS: HNE promotes the release of TF+ microvesicles from perivascular cells into the circulation. HNE-induced increased TF activity contributes to intravascular coagulation and inflammation.


Assuntos
Aldeídos/toxicidade , Micropartículas Derivadas de Células/efeitos dos fármacos , Inflamação/induzido quimicamente , Estresse Oxidativo , Tromboplastina/metabolismo , Trombose/induzido quimicamente , Actinas/metabolismo , Aldeídos/administração & dosagem , Animais , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Citocinas/sangue , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/sangue , Mediadores da Inflamação/sangue , Masculino , Camundongos Endogâmicos C57BL , Trombina/metabolismo , Trombose/sangue
12.
Int J Mol Sci ; 23(24)2022 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-36555120

RESUMO

Inflammation is a biological response of the immune system to various insults, such as pathogens, toxic compounds, damaged cells, and radiation. The complex network of pro- and anti-inflammatory factors and their direction towards inflammation often leads to the development and progression of various inflammation-associated diseases. The role of small non-coding RNAs (small ncRNAs) in inflammation has gained much attention in the past two decades for their regulation of inflammatory gene expression at multiple levels and their potential to serve as biomarkers and therapeutic targets in various diseases. One group of small ncRNAs, microRNAs (miRNAs), has become a key regulator in various inflammatory disease conditions. Their fine-tuning of target gene regulation often turns out to be an important factor in controlling aberrant inflammatory reactions in the system. This review summarizes the biogenesis of miRNA and the mechanisms of miRNA-mediated gene regulation. The review also briefly discusses various pro- and anti-inflammatory miRNAs, their targets and functions, and provides a detailed discussion on the role of miR-10a in inflammation.


Assuntos
MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação da Expressão Gênica , Biomarcadores , Inflamação/metabolismo
13.
Am J Respir Cell Mol Biol ; 64(4): 477-491, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33600743

RESUMO

Streptococcus pneumoniae is the leading cause of hospital community-acquired pneumonia. Patients with pneumococcal pneumonia may develop complicated parapneumonic effusions or empyema that can lead to pleural organization and subsequent fibrosis. The pathogenesis of pleural organization and scarification involves complex interactions between the components of the immune system, coagulation, and fibrinolysis. EPCR (endothelial protein C receptor) is a critical component of the protein C anticoagulant pathway. The present study was performed to evaluate the role of EPCR in the pathogenesis of S. pneumoniae infection-induced pleural thickening and fibrosis. Our studies show that the pleural mesothelium expresses EPCR. Intrapleural instillation of S. pneumoniae impairs lung compliance and lung volume in wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. Intrapleural S. pneumoniae infection induces pleural thickening in wild-type mice. Pleural thickening is more pronounced in EPCR-overexpressing mice, whereas it is reduced in EPCR-deficient mice. Markers of mesomesenchymal transition are increased in the visceral pleura of S. pneumoniae-infected wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. The lungs of wild-type and EPCR-overexpressing mice administered intrapleural S. pneumoniae showed increased infiltration of macrophages and neutrophils, which was significantly reduced in EPCR-deficient mice. An analysis of bacterial burden in the pleural lavage, the lungs, and blood revealed a significantly lower bacterial burden in EPCR-deficient mice compared with wild-type and EPCR-overexpressing mice. Overall, our data provide strong evidence that EPCR deficiency protects against S. pneumoniae infection-induced impairment of lung function and pleural remodeling.


Assuntos
Receptor de Proteína C Endotelial/deficiência , Pulmão/metabolismo , Pleura/metabolismo , Derrame Pleural/metabolismo , Pleurisia/metabolismo , Pneumonia Pneumocócica/metabolismo , Streptococcus pneumoniae/patogenicidade , Animais , Carga Bacteriana , Células Cultivadas , Modelos Animais de Doenças , Receptor de Proteína C Endotelial/genética , Feminino , Fibrose , Interações Hospedeiro-Patógeno , Humanos , Pulmão/microbiologia , Pulmão/patologia , Pulmão/fisiopatologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Pleura/microbiologia , Pleura/patologia , Derrame Pleural/microbiologia , Derrame Pleural/patologia , Derrame Pleural/fisiopatologia , Pleurisia/microbiologia , Pleurisia/patologia , Pleurisia/fisiopatologia , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/patologia , Pneumonia Pneumocócica/fisiopatologia
14.
Blood ; 134(7): 645-655, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31262782

RESUMO

Tissue factor (TF) is a cofactor for factor VIIa and the primary cellular initiator of coagulation. Typically, most TF on cell surfaces exists in a cryptic coagulant-inactive state but are transformed to a procoagulant form (decryption) following cell activation. Our recent studies in cell model systems showed that sphingomyelin (SM) in the outer leaflet of the plasma membrane is responsible for maintaining TF in an encrypted state in resting cells, and the hydrolysis of SM leads to decryption of TF. The present study was carried out to investigate the relevance of this novel mechanism in the regulation of TF procoagulant activity in pathophysiology. As observed in cell systems, administration of adenosine triphosphate (ATP) to mice enhanced lipopolysaccharide (LPS)-induced TF procoagulant activity in monocytes. Treatment of mice with pharmacological inhibitors of acid sphingomyelinase (ASMase), desipramine and imipramine, attenuated ATP-induced TF decryption. Interestingly, ASMase inhibitors also blocked LPS-induced TF procoagulant activity without affecting the LPS-induced de novo synthesis of TF protein. Additional studies showed that LPS induced translocation of ASMase to the outer leaflet of the plasma membrane and reduced SM levels in monocytes. Studies using human monocyte-derived macrophages and endothelial cells further confirmed the role of ASMase in LPS- and cytokine-induced TF procoagulant activity. Overall, our data indicate that LPS- or cytokine-induced TF procoagulant activity requires the decryption of newly synthesized TF protein by ASMase-mediated hydrolysis of SM. The observation that ASMase inhibitors attenuate TF-induced coagulation raises the possibility of their therapeutic use in treating thrombotic disorders associated with aberrant expression of TF.


Assuntos
Citocinas/metabolismo , Lipopolissacarídeos/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Tromboplastina/metabolismo , Trombose/metabolismo , Animais , Coagulação Sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Trombina/metabolismo
15.
Arterioscler Thromb Vasc Biol ; 40(5): 1275-1288, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32212848

RESUMO

OBJECTIVE: Recent studies showed that FVIIa (factor VIIa), upon binding to EPCR (endothelial cell protein C receptor), elicits endothelial barrier stabilization and anti-inflammatory effects via activation of PAR (protease-activated receptor)-1-mediated signaling. It is unknown whether FVIIa induces PAR1-dependent cytoprotective signaling through cleavage of PAR1 at the canonical site or a noncanonical site, similar to that of APC (activated protein C). Approach and Results: Mouse strains carrying homozygous R41Q (canonical site) or R46Q (noncanonical site) point mutations in PAR1 (QQ41-PAR1 and QQ46-PAR1 mice) were used to investigate in vivo mechanism of PAR1-dependent pharmacological beneficial effects of FVIIa. Administration of FVIIa reduced lipopolysaccharide-induced inflammation, barrier permeability, and VEGF (vascular endothelial cell growth factor)-induced barrier disruption in wild-type (WT) and QQ46-PAR1 mice but not in QQ41-PAR1 mice. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1, and QQ46-PAR1 mice showed that FVIIa activation of Akt (protein kinase B) in endothelial cells required R41 cleavage site in PAR1. Our studies showed that FVIIa cleaved endogenous PAR1 in endothelial cells, and FVIIa-cleaved PAR1 was readily internalized, unlike APC-cleaved PAR1 that remained on the cell surface. Additional studies showed that pretreatment of endothelial cells with FVIIa reduced subsequent thrombin-induced signaling. This process was dependent on ß-arrestin1. CONCLUSIONS: Our results indicate that in vivo pharmacological benefits of FVIIa in mice arise from PAR1-dependent biased signaling following the cleavage of PAR1 at the canonical R41 site. The mechanism of FVIIa-induced cytoprotective signaling is distinctly different from that of APC. Our data provide another layer of complexity of biased agonism of PAR1 and signaling diversity.


Assuntos
Anti-Inflamatórios/administração & dosagem , Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Fator VIIa/administração & dosagem , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Receptor PAR-1/metabolismo , Animais , Modelos Animais de Doenças , Endocitose , Células Endoteliais/metabolismo , Endotoxinas , Feminino , Homozigoto , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Transgênicos , Pneumonia/induzido quimicamente , Pneumonia/genética , Pneumonia/metabolismo , Mutação Puntual , Receptor PAR-1/genética , Transdução de Sinais
16.
Blood ; 131(21): 2379-2392, 2018 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-29669778

RESUMO

Recent studies show that endothelial cell protein C receptor (EPCR) interacts with diverse ligands, in addition to its known ligands protein C and activated protein C (APC). We showed in earlier studies that procoagulant clotting factor VIIa (FVIIa) binds EPCR and downregulates EPCR-mediated anticoagulation and induces an endothelial barrier protective effect. Here, we investigated the effect of FVIIa's interaction with EPCR on endothelial cell inflammation and lipopolysaccharide (LPS)-induced inflammatory responses in vivo. Treatment of endothelial cells with FVIIa suppressed tumor necrosis factor α (TNF-α)- and LPS-induced expression of cellular adhesion molecules and adherence of monocytes to endothelial cells. Inhibition of EPCR or protease-activated receptor 1 (PAR1) by either specific antibodies or small interfering RNA abolished the FVIIa-induced suppression of TNF-α- and LPS-induced expression of cellular adhesion molecules and interleukin-6. ß-Arrestin-1 silencing blocked the FVIIa-induced anti-inflammatory effect in endothelial cells. In vivo studies showed that FVIIa treatment markedly suppressed LPS-induced inflammatory cytokines and infiltration of innate immune cells into the lung in wild-type and EPCR-overexpressing mice, but not in EPCR-deficient mice. Mechanistic studies revealed that FVIIa treatment inhibited TNF-α-induced ERK1/2, p38 MAPK, JNK, NF-κB, and C-Jun activation indicating that FVIIa-mediated signaling blocks an upstream signaling event in TNFα-induced signaling cascade. FVIIa treatment impaired the recruitment of TNF-receptor-associated factor 2 into the TNF receptor 1 signaling complex. Overall, our present data provide convincing evidence that FVIIa binding to EPCR elicits anti-inflammatory signaling via a PAR1- and ß-arrestin-1 dependent pathway. The present study suggests new therapeutic potentials for FVIIa, which is currently in clinical use for treating bleeding disorders.


Assuntos
Receptor de Proteína C Endotelial/metabolismo , Fator VIIa/metabolismo , Inflamação/metabolismo , Receptor PAR-1/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Receptor de Proteína C Endotelial/genética , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/genética , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/imunologia , Camundongos , Receptor PAR-1/genética , Fator de Necrose Tumoral alfa/metabolismo , beta-Arrestinas/metabolismo
17.
Curr Opin Hematol ; 25(3): 219-226, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29461258

RESUMO

PURPOSE OF REVIEW: Endothelial cell protein C receptor (EPCR), a transmembrane glycoprotein present on the surface of endothelial cells and other cell types, is an essential component of the protein C (PC) anticoagulant system. EPCR is also shown to play a critical role in mediating activated protein C (APC)-induced cytoprotective signaling. The purpose of this review is to outline the mechanisms of EPCR-dependent cell signaling and discuss recent findings made in this area. RECENT FINDINGS: Recent studies showed that the cleavage of protease-activated receptor (PAR)1 at a noncanonical site by APC-EPCR or the canonical site by thrombin when PC occupies EPCR induces ß-arrestin-2-mediated biased cytoprotective signaling. Factor VIIa binding to EPCR is also shown to induce the cytoprotective signaling. EPCR is found to be a reliable surface marker for identifying human hematopoietic stem cells in culture. EPCR, binding to diverse ligands, is thought to play a role in the pathogenesis of severe malaria, immune functions, and cancer by either blocking the APC-mediated signaling or by mechanisms that are yet to be elucidated. SUMMARY: Recent studies provide a mechanistic basis to how EPCR contributes to PAR1-mediated biased signaling. EPCR may play a role in influencing a wide array of biological functions by binding to diverse ligands.


Assuntos
Células Endoteliais/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Fator VIIa/metabolismo , Proteína C/metabolismo , Receptor PAR-1/metabolismo , Transdução de Sinais , Animais , Células Endoteliais/patologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Malária/metabolismo , Malária/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , beta-Arrestina 2/metabolismo
18.
Arterioscler Thromb Vasc Biol ; 33(7): 1601-11, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23640483

RESUMO

OBJECTIVE: 4-hydroxy-2-nonenal (HNE) is one of the major aldehydes formed during lipid peroxidation and is believed to play a role in the pathogenesis of atherosclerosis. The objective of the present study is to investigate the effect of HNE on tissue factor (TF) procoagulant activity expressed on cell surfaces. APPROACH AND RESULTS: TF activity and antigen levels on intact cells were measured using factor Xa generation and TF monoclonal antibody binding assays, respectively. Exposure of phosphatidylserine on the cell surface was analyzed using thrombin generation assay or by binding of a fluorescent dye-conjugated annexin V. 2',7'-dichlorodihydrofluorescein diacetate was used to detect the generation of reactive oxygen species. Our data showed that HNE increased the procoagulant activity of unperturbed THP-1 cells that express traces of TF antigen, but had no effect on unperturbed endothelial cells that express no measurable TF antigen. HNE increased TF procoagulant activity but not TF antigen of both activated monocytic and endothelial cells. HNE treatment generated reactive oxygen species, activated p38 mitogen-activated protein kinase, and increased the exposure of phosphatidylserine at the outer leaflet in THP-1 cells. Treatment of THP-1 cells with an antioxidant, N-acetyl cysteine, suppressed the above HNE-induced responses and negated the HNE-mediated increase in TF activity. Blockade of p38 mitogen-activated protein kinase activation inhibited HNE-induced phosphatidylserine exposure and increased TF activity. CONCLUSIONS: HNE increases TF coagulant activity in monocytic cells through a novel mechanism involving p38 mitogen-activated protein kinase activation that leads to enhanced phosphatidylserine exposure at the cell surface.


Assuntos
Aldeídos/farmacologia , Monócitos/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tromboplastina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Citocinas/farmacologia , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ativação Enzimática , Fator Xa/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Monócitos/enzimologia , Monócitos/imunologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Trombina/metabolismo , Fatores de Tempo
19.
J Thromb Haemost ; 22(2): 441-454, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37926194

RESUMO

BACKGROUND: Factor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa-released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent cytoprotective responses. OBJECTIVES: To investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. METHODS: Activation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs. RESULTS: FVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa-driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa-EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM-/- mice against lipopolysaccharide-induced inflammation and vascular leakage. CONCLUSION: Our data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.


Assuntos
Vesículas Extracelulares , MicroRNAs , Camundongos , Animais , Fator VIIa/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais , Lipopolissacarídeos/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo
20.
Blood ; 117(11): 3199-208, 2011 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-21252088

RESUMO

Recent studies have shown that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C, but the physiologic significance of this interaction is unclear. In the present study, we show that FVIIa, upon binding to EPCR on endothelial cells, activates endogenous protease activated receptor-1 (PAR1) and induces PAR1-mediated p44/42 mitogen-activated protein kinase (MAPK) activation. Pretreatment of endothelial cells with FVIIa protected against thrombin-induced barrier disruption. This FVIIa-induced, barrier-protective effect was EPCR dependent and did not involve PAR2. Pretreatment of confluent endothelial monolayers with FVIIa before thrombin reduced the development of thrombin-induced transcellular actin stress fibers, cellular contractions, and paracellular gap formation. FVIIa-induced p44/42 MAPK activation and the barrier-protective effect are mediated via Rac1 activation. Consistent with in vitro findings, in vivo studies using mice showed that administration of FVIIa before lipopolysaccharide (LPS) treatment attenuated LPS-induced vascular leakage in the lung and kidney. Overall, our present data provide evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier-protective effect. These findings are novel and of great clinical significance, because FVIIa is used clinically for the prevention of bleeding in hemophilia and other bleeding disorders.


Assuntos
Antígenos CD/metabolismo , Células Endoteliais/metabolismo , Fator VIIa/metabolismo , Receptor PAR-1/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Animais , Permeabilidade Capilar/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Receptor de Proteína C Endotelial , Ativação Enzimática/efeitos dos fármacos , Fator VIIa/administração & dosagem , Fator VIIa/farmacologia , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Trombina/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA